Slutredovisning till Partnerskap Alnarp för 2012-2013

Slutredovisning till Partnerskap Alnarp för 2012-2013
Förbättra rotbildningsförmågan hos svårrotade mikroförökade växtslag
Projektansvarig: Margareta Welander, Professor SLU Alnarp
Medsökande: Elisabeth Nilsson, Verksamhetsledare på Elitplantstationen
Mikroförökning av Aristolocia manchuriensis
Skottproduktion, rotning och acklimatisering
Margareta Welander och Tatiana Kuznetsova
Institutionen för växtförädling och bioteknik, SLU, Alnarp
1
Sammanfattning
Syftet med projektet har varit att förbättra rotbildningsförmågan samt etablering i jord
hos svårförökade växter eftertraktade av plantskolorna. Eftersom piprankan
(Aristolochia manchuriensis) visat sig vara det mest svårförökade och efterfrågade
växtslaget har forskningen koncentrerats till detta växtslag.
Mikroförökade skott av Aristolochia manchuriensis har en rotningsförmåga på 1020% vilket gjort att efterfrågan på plantor varit mycket större än vad Elitplantstationen
inte kunnat leverera. Institutionen för växtförädling och bioteknik, SLU Alnarp samt
företaget vitroform i Årslev,Danmark har bedrivit intensiva studier under mer än 1 års
tid på skottproduktion och rotning av Aristolochia manchuriensis medan
Elitplantstation stått för plantetablering.
Försöken utförda vid SLU finns redovisade i 2 bilagor.
1. Improvement of rooting ability and ex vitro acclimatization of difficult to root
micrpropagated Aristolochia manchuriensis plants
2. Light impact on shoot growth, quality and rooting of Aristolochia
manchuriensis plants
Slutsatser
Skottproduktion
Pipranka har för första gången förökats i bioreaktorer i flytande näringslösning
baserat på Temporary Immersion System( ebb och flod). Skott av pipranka var
mycket känsliga för vitrifiering. Genom att minska BAP koncentration, badtid ( antal
gånger och tid skotten badar i näringslösningen) samt ökad tid för ventilering av
odlingskärl minskades vitrifieringsgraden och 60 % av skotten var gröna och friska.
Mikroförökade skott av pipranka
I bioreaktor
i bioreaktorbioreaktor
2
Rotning
Rotningsförsök utfördes på mikroförökade skott från både bioreaktorer och halvfast
agar medium (semisolid medium). Rotning i bioreaktorer misslyckades på grund av
vitrifiering. Rotning i agar medium innehållande 1/3 Lepoivre medium plus extra
MgSO4 och 1mg/l IAA resulterade i 70% rotning. Tyvärr brunfärgades många blad
vilket visar på överkänslighet mot auxin.
Rotade plantor på 1/3 Lepoivre medium med extra MgSO4 och 1mg/l IAA
Reviderat rotningsmedium baserat på näringsanalys av bladen resulterade inte i
ökad rotbildning.
Ljusförsök
Flera undersökningar har visat på positiva effekter av LED ( light emitting diodes) ljus
på tillväxt och rotutveckling hos olika växtslag. I våra försök användes vitt, blått och
rött, enbart blått och rött LED ljus:
3
Olika ljusbehandlingar hade ingen effekt på rotbildning hos pipranka till skillnad från
andra växtslag.
Försök utförda av Lars Sommer ägare till företaget vitroform i Årslev,Danmark.
Svårigheter med vitrifiering av pipranka noterades även av Lars Sommer. Detta ledde
till att agarmängden ökades både i förökningsmediet och i rotningsmediet. Liksom i
våra försök visade det sig att ammoniumjoner och tillväxthormoner i rotningsmediet
hämmade tillväxten. Genom att införa en ny fas mellan skottproduktion och rotning
med låg BAP koncentration och därefter tillsätta aktivt kol i rotningsmediet har vi nu
problemet under rimlig kontroll och acceptabel etablering i jord på Eliplantstationen.
4
Improvement of rooting ability
and ex vitro acclimatization
of difficult to root micropropagated
Aristolochia manshuriensis plants
Ph.D. T. Kuznetsova
Supervisor: Prof. M. Welander
SLU, Swedish University of Agricultural Sciences,
Department of Plant Breeding and Biotechnology
Alnarp, 2012
1
Contents
Introduction .................................................................................................................................................. 3
Materials and methods .............................................................................................................................. 4
Plant material .......................................................................................................................................... 4
Micropropagation of shoots on semi-solid media .............................................................................. 4
Micropropagation of shoots in liquid media in TIS ............................................................................ 4
Modification of immersion and ventilation cycles in TIS ............................................................... 5
In vitro rooting of shoots on semi-solid media and by dipping technique ............................................... 6
In vitro rooting of shoots in liquid media in TIS .................................................................................... 8
Ex vitro rooting of shoots ....................................................................................................................... 8
Acclimatization of in vitro rooted shoots............................................................................................10
Chemical analysis of shoots for development of rooting media ....................................................11
Results and discussion ............................................................................................................................12
Micropropagation of shoots on semi-solid media ............................................................................12
Micropropagation of shoots in liquid media in TIS ..........................................................................12
Modification of immersion and ventilation cycles in TIS .............................................................13
In vitro rooting of shoots in semi-solid media and by dipping technique...........................................15
In vitro rooting of shoots in liquid media in TIS ................................................................................18
Ex vitro rooting of shoots .....................................................................................................................20
Acclimatization of in vitro rooted shoots............................................................................................22
Chemical analysis for development of rooting media .....................................................................24
Conclusion .................................................................................................................................................26
Literature ....................................................................................................................................................28
2
Introduction
The ornamental plant Aristolochia manshuriensis Kom. is difficult to propagate
either generatively by seeds or vegetatively by layerings or cuttings (Hansen,
1999). An alternative way of propagation could be in vitro tissue culture with
production of many individuals from a single explant. It is known from the
literature (Svensson, 2000, Hedman, 2005) that the rooting ability of shoots in vitro
from A. manshuriensis plants is very poor and that they are extremely sensitive to
ex vitro acclimatization. In order to scale up plant production of A. manshuriensis
in vitro a new culture vessel system (Plantform bioreactor: www.plantform.se)
based on temporary immersion system (TIS) was tested in comparison with semisolid agar medium. The Plantform bioreactor has been appropriate for large scale
propagation and rooting of many species (Rubus, Echinacea, Blueberry,
Clematis, Ensete).
The objective was to optimize a system for the in vitro production of green
vigorous rooted plants of Aristolochia manshuriensis by
1) Developing media and suitable conditions for propagation of plants in liquid
medium in Plantform bioreactor and semi-solid agar medium
2) Developing media for root initiation
3) Improving ex vitro acclimatization
3
Materials and methods
Plant material
Shoot cultures of Aristolochia manshuriensis Kom. kindly provided by Lars
Sommer (Vitroform, Denmark) was used in all experiments.
Micropropagation of shoots on semi-solid media
Two types of media were used for shoot production: Lep (Quoirin and Lepoivre,
1977) multiplication medium containing Lep macronutrients, MS (Murashige and
Skoog, 1962) micronutrients, MS vitamins and MS multiplication medium
containing MS macronutrients, MS micronutrients, MES buffer and LS
(Linsmayer and Skoog, 1965) vitamins. The media were supplemented with 0,15;
0,2 or 0,6 mg/l benzylaminopurine (BAP), 30 g/l sucrose dissolved in double
distilled water. Doubling of Fe concentration was tested as well. The pH was
adjusted to 5,5 in Lep and 5,7 in MS media with NaOH before autoclaving for 20
min at 120°C. All semi-solid media were gelled using 7 g/l Bacto agar. The
cultures were kept in a controlled environment chamber under a 16-h
photoperiod from cool white fluorescent tubes (F 96 T12 / CW / VHO) with
33 μmol m-2s-1 at 24/18±1ºC day/night temperatures. Shoots were subcultured
monthly by transferring nine 5 mm segments into 62,5 ml of fresh semi-solid
medium in 0,4 l plastic jars (Styrolux Thermoplastic).
Micropropagation of shoots in liquid media in TIS
Plantform bioreactor (3,75 l) was used for propagation and rooting experiments in
liquid media in TIS. The bioreactor and its different parts are described in the
website: www.plantform.se. Bioreactors (fig. 1 a) were connected to two pump
a
d
e
c
b
Fig. 1. Plantform bioreactors (a) connected to two
pumps (b, c) by means of silicone hoses (d).
Programmable timers (e) regulate the pumps.
4
systems P1 and P2 (fig. 1 b, c) by means of 60 cm silicone hoses (ID = 4 mm)
(fig. 1 d) and immersion and ventilation time programs were set in programmable
timers (fig. 1 e). The movement of liquid medium in the bioreactor is described in
figure 2. Figure 2 A shows the medium level when both air pumps are OFF. The
pressure applied by air pump P1 in the lower compartment pushes the medium and
it floods the basket with the plant material (fig. 2 B). Plants are immersed as long as
pressure from immersion pump P1 is applied. When pressure from P1 is relieved
and second air pump P2 starts to work the nutrients drains back to the inner
chamber through the holes in the basket (fig. 2 C) and headspace of the bioreactor
is ventilated (fig. 2 D). 500 ml of liquid propagation medium was used in each
bioreactor. 33 (five mm) shoots without leaves were placed in each bioreactor. The
media tested in the bioreactors were the same as for semi solid media except agar.
Hormone free medium were tested as well. Explants were subcultured for 4 weeks.
Modification of immersion and ventilation cycles in TIS
The duration (min) and frequency (number) of immersion and ventilation per day
was modified according to table 1. Standard program consisting of 4 min
immersion 2 times every 24 h and 4 min ventilation every hour from 8.00 till
20.00 was compared with 4 modifications for efficiency in improving the shoot
production of A. manshuriensis in TIS.
A
P1 – OFF
P2 – OFF
B
C
P1 – OFF
P2 – ON (after 4 min)
D
P1 – ON (after 1 min)
P2 – OFF
P1 – OFF
P2 – ON (after 5 min)
Fig. 2. Movement of liquid media in Plantform bioreactor: A – media
level when both air pumps are OFF; B – media floods the basket
with the plant material, when P1 is ON; C – media drains back
through the holes in the basket, when P1 is OFF and P2 is ON, D –
ventilation of headspace, when P2 is ON
5
Table 1. Modification of immersion and ventilation cycles for propagation in TIS
No
1.
2.
3.
4.
5.
Immersion
frequency
duration
2 times per 24 h
1 time per 24 h
6 times per 24 h
4 min
1 time per 33 h
1 time per 48 h
Ventilation
frequency
duration
4 min
10 min
Hourly
from 8.00
10 min
till 20.00
10 min
4 min
Abbreviation
of program
2/24+4’
1/24+10’
6/24+10’
1/33+10’
1/48+4’
Nine bioreactors were connected to two pumps (Hailea air pump ACO 9602, China,
7,2 l/min, 432 l/h) for each program combination. Duration and frequency of immersion
and ventilation were controlled with two programmable timers. Different concentrations
of BAP (0,15 and 0,6 mg/l) for shoot production were tested as well. Different
immersion and ventilation cycles were compared for their efficiency by evaluating the
percentage of hyperhydric shoots, healthy well growing shoots, healthy slowly growing
shoots and healthy not growing shoots. The height, weight of the shoots and number
of leaves produced were recorded also after 4 weeks of cultivation.
In vitro rooting of shoots on semi-solid media and by dipping technique
Healthy single shoots (2 cm or more) were excised from multiple shoot clusters
growing on propagation semi-solid medium and placed on semi-solid rooting media.
The basic culture media for in vitro rooting of A. manshuriensis shoots were either 1/3
strength MS or 1/3 strength Lep supplemented with 3% sucrose. Different
concentrations of the auxins IAA (indole-3-acetic acid), NAA (1-naphthaleneacetic
acid), 2,4-dichlorophenoxyacetic acid (2,4-D) and IBA (indole-3-butyric acid) were
tested for rooting. The auxins were included individually or together with cytokinins
(BAP (6-benzylaminopurine) and kinetin) in semi-solid media (Table 2).
Occasionally full and 1/2 strength of rooting media, lack of NH4NO3 and extra
600 mg/l MgSO4 were tested as well. Splitting of basal ends of shoots was also
tested. Plants on rooting media had dark treatment during the first 5 days. In some
media 2 g/l of activated charcoal was added instead of 5 days dark treatment.
Rooting by dipping technique was performed by using 2,5-cm-long shoots
exposed to 250 mg/l IBA or IAA solutions during 2 min for root induction. To
increase area of surface absorption basal end of the shoot was cut 1 to 2 mm up
the stem with sharp scalpel and then dipped in hormone solution. Thereafter,
explants were transferred to Lep or MS hormone-free basal medium with 2 g/l
charcoal for root initiation and then after 10 days on Lep or MS medium with
0,15 mg/l BAP (low BAP media). Before root induction occasionally low BAP
media pre-treatment was also tested. The shoots were cultivated on low BAP Lep
or MS media for 4 weeks two times to leach out the cytokinin from the shoots and
6
Table 2. Different types and concentrations of hormones in Lep or MS semi-solid media
for root initiation
Type of media
Auxin in high concentration
½ Lep
1/3 Lep
1/3 MS
Auxin in high concentration during short period
Dipping + Low BAP MS
Dipping + Low BAP Lep
Dipping + Low BAP MS
Dipping + Low BAP Lep
Low BAP(x2) + Dipping + low BAP MS
Low BAP(x2) + Dipping + low BAP Lep
Low BAP(x2) + Dipping + low BAP MS
Low BAP(x2) + Dipping + low BAP Lep
Combination of auxin and cytokinin
1/3 MS
1/3 MS
1/3 MS
1/3 MS
1/3 Lep
1/3 Lep
1/3 Lep + charcoal
MS
MS
Auxin in low concentration
1/3 Lep
1/3 MS
1/3 Lep
1/3 MS
1/3 Lep
1/3 Lep + charcoal
Lep NH4NO3 free + charcoal
Lep NH4NO3 free + charcoal
MS NH4NO3 free
1/3 Lep + MgSO4
1/3 MS + MgSO4
Type
Hormone
Concentration, mg/l
IAA
IAA
IAA
20
10
10
IAA
IAA
IBA
IBA
IAA
IAA
IBA
IBA
250
250
250
250
250
250
250
250
IBA + kinetin
IBA + BAP
IBA + NAA
kinetin + NAA
NAA + BAP
IAA + BAP
IAA + BAP
IAA + BAP
IAA + BAP
2+2,5
1,5+2
2+2
2,5+2
1+0,1
10+1
1+0,1
1+0,1
0,5+0,05
IBA
IBA
2,4-D
2,4-D
NAA
IAA
IAA
IAA
IAA
IAA
IAA
1
1
1
1
1
1
1
5
1
1
1
to elongate plants. At the end of the 4-week of multiplication cycle, newly
developed 2,5-cm-long shoots were harvested and rooted as above.
On rooting media the percentage of rooted shoots, callus growth and green
shoots were recorded. Also plants with and without leaves, rooted plant with and
without leaves, number of roots per plants and length of roots were calculated.
All data were collected after 4 weeks.
7
In vitro rooting of shoots in liquid media in TIS
The same type of shoots as used for rooting on semi-solid media was placed in
Plantform bioreactor in liquid rooting media. The rooting media tested in TIS are
shown in table 3. In order to get dark conditions during first 5 days Plantform vessels
were covered with foil and connected to pumps.
Table 3. Different types and concentrations of hormones in Lep or MS liquid media for
root initiation
Type of media
Type
Auxin in high concentration
1/3 Lep
IAA
½ Lep
IAA
1/3 MS
IAA
1/3 Lep
IAA
1/3 MS
IAA
Auxin in high concentration during short period
1/3 Lep
IAA
1/3 MS
IAA
Combination of auxin and cytokinin
1/3 MS
IBA + kinetin
1/3 MS
IBA + BAP
1/3 MS
IBA + NAA
1/3 MS
kinetin + NAA
1/3 MS
IAA + BAP
Auxin in low concentration
1/3 Lep
2,4-D
1/3 MS
2,4-D
1/3 Lep
IBA
1/3 MS
IBA
1/3 Lep
IAA
1/3 MS
IAA
1/3 Lep + MgSO4
IAA
1/3 MS + MgSO4
IAA
Lep NH4NO3-free
IAA
MS NH4NO3-free
IAA
Hormone
Concentration, mg/l
20
20
20
10
10
20 (for 3 days)/0
20 (for 3 days)/0
2+2,5
1,5+2
2+2
2,5+2
1+0,1
1
1
1
1
1
1
1
1
1
1
Ex vitro rooting of shoots
Ex vitro rooting was performed in sterile boxes (fig. 3) with peat or in none sterile
pots (fig. 4) with mixture of planting soil and perlite. The sterile box (fig. 3 A)
consisted of a) a transparent box (190x290x85 mm) with b) a white tray inside. The
tray consisted of 48 individual cells with a hole at the bottom. Each cell contained
peat plugs (c) wrapped with plastic net bags. 3 cm shoots (d) obtained in vitro were
placed individually (fig. 3 B) into peat plugs supplemented with sterile liquid rooting
medium. Thereafter the box was covered with a ventilated sterile plastic bag (f) and
sealed. Plastic bags had filter stripes (e) for ventilation of plants inside the box to
8
A
a
B
b
e
c
f
d
Fig. 3. A sterile soil box (A) for ex vitro rooting of shoots covered with ventilated plastic bag (B):
a) a transparent box, b) a 48 cells white tray, c) peat plugs wrapped with plastic net bags, d)
shoots, e) plastic bag with f) filters for ventilation
improve acclimatization of plants and prepare for ex vitro conditions. Three different
rooting media were used namely 1/3 strength of Lep medium supplemented with
three different concentrations of IAA (5, 10, 20 mg/l). A volume of 0,5 l of sterile liquid
rooting medium was poured per tray to wet the peat. All operations with the box were
done under sterile conditions in a laminar flow bench. During the first week the box
were kept in a growth chamber and then placed in green house.
The pots (55x55x70 mm) (fig. 4) used for rooting were filled with none sterile
mixture of planting soil and perlite (3:2). The rooting medium was added to the
soil only once before planting of 3 cm shoots and afterwards irrigated manually
when needed. 2 l medium was used per 7,5 l of soil mixture. The medium used
consisted of 1/3 strength of Lep rooting medium supplemented with either 5, 10,
or 20 mg/l of IAA. 1/3 strength of MS medium with 10 mg/l IAA was tested
additionally. Only water added to the planting mixture was used as control.
We also tested dipping of shoots in 250mg/l IAA solution for 20 minutes and
then placing them in soil wetted with water. Minimum 40 shoots were used per
A
B
Fig. 4. None sterile pots with mixture of planting Fig. 5. Pots covered with plastic film (A) or
placed in styrofoam boxes with glass lid (B)
soil and perlite for ex vitro rooting of shoots
9
treatment. The pots with plants were covered with plastic film (fig. 5 A) or placed
into styrofoam box (fig. 5 B) (350x850x350, thickness of the walls – 5 cm)
covered by a transparent glass lid. The pots and boxes were placed in green
house. The temperature inside the box was 26°C. After ex vitro rooting the
percentage of survived plants was recorded after one and two months.
Acclimatization of in vitro rooted shoots
Rooted in vitro plantlets were carefully removed from the semi-solid or liquid
media, washed thoroughly with water and potted in a mixture of planting soil and
perlite (3:2) or peat. Trays (300x800x120) (fig. 6 A), pots (55x55x70) (fig. 4) or
peat plugs (OD=35 mm, high – 40 mm) (fig. 6 B) were used for ex vitro
A
B
Fig. 6. Trays with a mixture of planting soil and perlite (A) and peat plugs (B) for acclimatization
of in vitro rooted shoots
acclimatization of in vitro rooted shoots. All plants were covered with transparent
plastic cups (fig. 7 A) to maintain high humidity around plants for the first 2 weeks
and later with floating row cover (fig. 7 B) which was sprayed periodically to
increase humidity and avoid desiccation of plants. After one and two months ex
vitro the percentage of survived plants was recorded.
A
B
Fig. 7. In vitro rooted plants covered with transparent plastic cups (A) during first 2 weeks and
later with floating row cover (B)
10
Chemical analysis of shoots for development of rooting media
Healthy green leaves picked from vigorous, nearly mature, ca 1 m tall plants (fig. 8)
grown in the field at Balsgård and 2-3 cm in vitro shoots (fig. 9) were taken for
chemical analysis performed by LMI (Lennart Månsson International). Fe, Al, Mo,
Cu, P, S, Zn, Cd, Mn, Ni, Na, Mg, Ca, K, Si were analyzed by ICP and total N by
elemental analysis using Vario Mac. Results of chemical analysis of shoots were
used for further development of a new rooting medium for growth of vigorous rooted
Aristolochia manshuriensis plants in vitro.
Fig. 8. Field grown A. manshuriensis plants
Fig. 9. In vitro grown A. manshuriensis shoots
11
Results and discussion
Micropropagation of shoots on semi-solid media
Shoots cultured on Lep semi-solid medium supplemented with 0,2 mg/l BAP
were slightly hyperhydrified. Occasionally the shoots were fragile, swelled glassy
and with curled bottom leaves. Moreover the leave blades of plants cultured on
growth medium were lighter than the veins. After decreasing concentration of
BAP to 0,15 mg/l, the cultured shoots became healthy, but multiplication rate
decreased from 5-6 to 1-3. Doubling of Fe concentration in medium did not
improve the green color of the leaves.
Micropropagation of shoots in liquid media in TIS
A. manshuriensis, as well as other members of the family Aristolochiaceae, has
not been propagated in liquid media in TIS before. Thus, the aim was to develop
suitable conditions for propagation in TIS using Plantform bioreactor. Figure 10
shows the results of different media combinations using TI program 2/24+4’ (table
1). On MS medium with 0,6 mg/l BAP the shoots were healthy but bottom leaves
were abnormal and strongly hyperhydrified (fig. 10 B). On Lep medium
supplemented with 0,2 mg/l BAP 100% of plants became hyperhydrified (fig. 10 C)
and on hormone free medium plants were not vitrified but did not grow (fig. 10 D).
A
B
C
D
Fig. 10. Shoots cultivated in 2/24+4’ TIS program on different media: at 0 days (A), in 45 days on MS
media (B), in 30 days on Lep media (C), in 30 days on hormone free MS media (D)
12
Modification of immersion and ventilation cycles in TIS
% of shoots
100
Shoots cultivated on MS medium
hyperhydric grow small not grow
with 0,15 mg/l BAP under 5
80
different ventilation and immersion
60
programs
(table
1)
showed
differences in shoot appearance
40
(fig. 11). In 2/24+4’ program 20% of
20
the shoots were healthy, 50% did
0
not
grow
and
30%
were
2/24+4' 1/24+10' 6/24+10' 1/33+10' 1/48+4'
hyperhydrified. In 1/24+10’ and
Immersion program
6/24+10’ programs, 48% or 42% of Fig. 11. Percentage of shoots with different
the shoots were healthy and well appearance cultivated on MS media with 0,15 mg/l
growing whereas 16 % were BAP in different immersion programs.
hyperhydrified. In 1/33+10’ program Legend description: hyperhydric – hyperhydric shoots, grow
only 3% of shoots became – healthy well growing shoots, small – healthy slowly
growing shoots, not grow – healthy not growing shoots
hyperhydric, but the percentage of
healthy well growing shoots was not higher than in other programs. The use of
1/48+4’ program led to the production of shoots without hyperhydricity but shoots
cultivated in 1/33+10’ and 1/48+4’ programs were pail (fig. 12 A, B) whereas in
1/24+10’ and 6/24+10’ programs shoot were greener (fig. 12 C, D).
A
B
C
D
Fig. 12. Shoots cultivated on MS medium with 0,15 mg/l BAP in different immersion programs
in TIS: 1/33+10’ (A), 1/48+4’ (B), 1/24+10’ (C) and 6/24+10’ (D)
13
Table 4 shows that in 1/24+10’ and 6/24+10’ programs shoot growth was better
and the height of healthy shoots was higher (9-12 mm) than in 1/33+10’ and
1/48+4’ programs (7 mm). The number of leaves per shoot in 1/24+10’ or
6/24+10’ programs (2-3) was also higher than in 1/33+10’ and 1/48+4’ programs
(1-2), as well as, shoot weight (0,17-0,20 g and 0,06 g respectively), leave area
and petiole growth (data not shown). The shoots height of hyperhydric shoots in
all studied programs were higher and they characterized by lager number of
leaves and fresh weight compared to normal healthy shoots (table 4).
Table 4. Shoot height, leaf number per shoot and shoot weight after one month cultivation in
different immersion and ventilation programs (table 1) on MS medium with 0,15 mg/l BAP
Parameters
Shoot height,
mm
Leaf number
per shoot
Shoot weight, g
Quality of shoots
healthy
hyperhydrified
healthy
hyperhydrified
healthy
hyperhydrified
Immersion and ventilation programs
2/24+4’ 6/24+10’ 1/24+10’ 1/33+10’ 1/48+4’
10
9-12
9-12
7
7
25
12
10
20
–
2-3
2-3
2-3
1-2
1-2
4-5
3-4
3-4
3-4
–
0,165
0,200
0,170
0,063
0,060
0,850
0,670
0,470
0,350
–
The impact of BAP concentration in the different immersion programs showed that
growth of shoots decreased with increasing BAP concentration from 0,15 mg/l till
0,6 mg/l. In program 1/33+10’ on medium with 0,15 mg/l BAP 26% of shoots did
not grow and with 0,6 mg/l BAP – 65%. In 1/48+4’ program 9% of shoots did not
grow on medium with low BAP and 32% – with high BAP (fig. 13 A). However on
MS media growth increased with the decreasing of immersion frequency no matter
of BAP concentration. Nutrient compositions of media also impacted on growth of
plants in different immersion programs (fig. 13 B).
100
A
100
80
%of shoots,
% of shoots
80
B
60
40
60
40
20
20
0
0
0,15 BAP
0,6MS
BAP
1/33
1/33+10
’
hyperhydric
vitrified
grow
0,15 BAP
0,6 MS
BAP
1/48
1/48+4’
small
not grow
0,15 BAP
MS
0,15 BAP
Lep
1/24
1/24+10’
hyperhydric
vitrified
grow
0,15 BAP
MS
0,15 BAP
Lep
6/24
6/24+10’
small
not grow
Fig. 13. Percentage of shoots with different appearance cultivated in different immersion programs
on MS medium with 0,15 or 0,6 mg/l BAP (A) and on MS or Lep media with 0,15 mg/l BAP (B)
Legend description: hyperhydric – hyperhydric shoots, grow – healthy well growing shoots, small – healthy
slowly growing shoots, not grow – healthy not growing shoots
14
In vitro rooting of shoots in semi-solid media and by dipping technique
Table 5 shows the results on rooting, shoot growth and callus development on
semi solid media with different compositions. Shoots incubated in ½ Lep medium
with 20 mg/l of IAA did not form roots in vitro because of strong shoot decline. On
media with 10 mg/l of IAA roots were developed only on 1/3 Lep medium. The
Table 5. Rooting and shoot development on semi-solid rooting media
Green shoot growth: – none, ± little, + strong; callus development: – none, ± little, + strong
Type of media
Hormone
Green
%
Callus
shoot
of
rooted
Type
Concentration,
development
growth
plants
mg/l
Auxin in high concentration
½ Lep
IAA
20
1/3 Lep
IAA
10
1/3 MS
IAA
10
Auxin in high concentration during short period
Dipping + Low BAP MS
IAA
250
Dipping + Low BAP Lep
IAA
250
Dipping + Low BAP MS
IBA
250
Dipping + Low BAP Lep
IBA
250
Low BAP(x2) + Dipping + low
IAA
250
BAP MS
Low BAP(x2) + Dipping + low
IAA
250
BAP Lep
Low BAP(x2) + Dipping + low
IBA
250
BAP MS
Low BAP(x2) + Dipping + low
IBA
250
BAP Lep
Combination of auxin and cytokinin
1/3 MS
IBA + kinetin
2+2,5
1/3 MS
IBA + BAP
1,5+2
1/3 MS
IBA + NAA
2+2
1/3 MS
kinetin + NAA
2,5+2
1/3 Lep
NAA + BAP
1+0,1
1/3 Lep
IAA + BAP
10+1
1/3 Lep + charcoal
IAA + BAP
1+0,1
MS
IAA + BAP
1+0,1
MS
IAA + BAP
0,5+0,05
Auxin in low concentration
1/3 Lep
IBA
1
1/3 MS
IBA
1
1/3 Lep
2,4-D
1
1/3 MS
2,4-D
1
1/3 Lep
NAA
1
1/3 Lep + charcoal
IAA
1
Lep NH4NO3 free + charcoal
IAA
1
Lep NH4NO3 free + charcoal
IAA
5
MS NH4NO3 free
IAA
1
1/3 Lep + MgSO4
IAA
1
1/3 MS + MgSO4
IAA
1
–
–
–
+
+
+
0
42
0
+
+
+
+
–
–
–
–
0
0
0
0
+
–
0
+
–
0
+
–
0
+
–
0
–
+
–
–
–
±
±
±
+
–
+
–
–
±
+
+
+
+
0
0
0
0
0
0
8
11 (22*)
0
–
–
–
–
–
±
+
+
±
±
±
–
–
+
+
±
–
–
–
–
±
±
0
0
0 (0*)
0 (0*)
0
4
0
0
17
70
37
* – splitting of basal ends of the shoots
15
rooting was 42% but shoots were fragile, brown with callus and show
hyperhydricity (fig. 14). Rooting of shoots with continuous presence of exogenous
auxins in high concentration was not appropriate due to hyperhydricity. The
technique to provide exogenous auxin during short period for induction and
reducing auxin content during the expression stage – dipping technique – also did
not show any stimulation of rooting but shoots were green and vigorous (fig. 15).
Fig. 14. Development of rooted plants on
1/3 Lep media with 10 mg/l IAA
Fig. 15. Rooting of shoots by dipping in
250 mg/l IAA
Different combinations of auxin and cytokinin did not improve rooting, except for
medium with IAA (1 mg/l) together with BAP (0,1 mg/l), but percentage of rooted
plants was very low (8% on 1/3 Lep medium and 11% on MS medium).
Moreover, the shoots cultured for 4 weeks on the media with auxin together with
BAP (IBA+BAP and IAA+BAP) were green but plantlets formed large basal callus
(fig. 16). Splitting of shoots at the basal ends improved rooting of shoots on MS
media with IAA together with BAP and the percentage of rooted plants increased
from 11% till 22%. Neither increased or reduced concentrations of IAA and BAP
(10/1 and 0,5/0,05 mg/l) resulted in better rooting. Auxins (IBA, 2,4-D, NAA) alone
in low concentrations did not result in any rooting. However IAA in low
concentration (1 mg/l) stimulated rooting. Full-strength of MS medium without
NH4NO3 resulted in 17% rooting. The highest rooting percentage was obtained on
media supplemented with extra MgSO4 together with 1 mg/l IAA on both 1/3 Lep
(70%) and 1/3 MS (37%).
Fig. 16. Rooted plants cultured on 1/3 Lep media with 1 mg/l IAA and 0,1 mg/l BAP
16
The rooting on 1/3 Lep medium with extra MgSO4 together with 1 mg/l IAA started
earlier than on similar 1/3 MS medium (fig. 17). After 2,5 weeks 38% of plants
cultivated on 1/3 Lep medium produced roots whereas only 3% – on 1/3 MS medium.
The number of rooted plants on 1/3 Lep medium after 3,5 weeks were 57% and on
1/3 MS medium 31 %. After 4 weeks the percentage of rooted plants was almost twice
as high on 1/3 Lep (71%) than on 1/3 MS medium (37%). However on rooting medium
53% of all shoots cultivated on 1/3 Lep medium were without leaves and 66% on 1/3
MS medium (fig. 18). Rooted shoots looked different (fig. 19) and not all were vigorous
and green. 32% of rooted shoots cultured on 1/3 Lep and 18% on 1/3 MS declined
and dropped the leaves (fig. 20 A). However, 39% of shoots rooted on 1/3 Lep
medium kept leaves and formed roots at the same time, whereas only 18% on 1/3 MS
medium (fig. 20 A). 9% of plants on 1/3 Lep media and 10% on 1/3 MS were green
and vigorous and had well developed roots (fig. 19 a, fig. 20 B). Some plants were
partly green (18% on 1/3 Lep and around 3% on 1/3 MS media) (fig. 19 b, fig. 20 B).
Small number of plants (around 2%) were half green on 1/3 Lep media (fig. 19 c, fig.
20 B). 9% of rooted plants on 1/3 Lep media and 5% on 1/3 MS media were brown
with wilted leaves and had only green part of shoot left (fig. 19 d, fig. 20 B).
100
80
80
Lep
60
% of shoots
% of rooted shoots
100
MS
40
20
no leaves
with leaves
60
40
20
0
0
0
10
20
30
1/3 Lep media
Days
Fig. 17. Rooting within 30 days cultivation on
1/3 Lep and 1/3 MS media supplemented
with 1 mg/l IAA and extra MgSO4
a
b
1/3 MS media
Fig. 18. The percentage of shoots with or
without leaves after 4 weeks on 1/3 Lep or 1/3
MS media with 1 mg/l IAA and extra MgSO4
c
d
Fig. 19. The appearance of rooted shoots on 1/3 Lep medium with 1 mg/l IAA and extra
MgSO4: green plant (a), 2/3 green (b), ½ green (c), 1/3 green (d) (bar=1 cm)
17
100
100
% of different types of rooteed shoots
A
% of rooted shoots
80
rooted
plants
without leaves
without
leaves
with leaves
rooted
with leaves
60
38,6
40
18,4
20
31,8
18,4
B
80
60
40
20
0
0
1/3Lep
Lepmedium
media
1/3
1/3MS
MS medium
media
1/3
31,8
9,1
2,3
18,2
Green
2/3 green
1/2 green
1/3 green
no leaves
18,4
9,1
5,3
2,6
10,5
1/3
Lepmedium
media
1/3
Lep
1/3MS
MSmedium
media
1/3
Fig. 20. Percentage of rooted shoots with or without leaves (A) and percentage of different types
of rooted shoots (B) after 4 weeks on 1/3 Lep or 1/3 MS media with 1 mg/l IAA and extra MgSO4
The maximum number of roots developed per plants on 1/3 Lep medium within 4
weeks was 14, and on 1/3 MS medium – 11. The length of roots were also higher
on 1/3 Lep than on 1/3 MS media, 20 mm and 12 mm respectively, whereas
mean length was 8,7 mm on 1/3 Lep medium and 5,4 mm on 1/3 MS medium.
In vitro rooting of shoots in liquid media in TIS
The biggest problem during rooting of shoots in liquid media was hyperhydricity.
The shoots (fig. 21 A) cultured on rooting MS or Lep media in TIS in 2/24+4’
A
B
C
D
Fig. 21. Development of shoots on rooting liquid media with different concentrations of IAA:
in 0 days (A) and in 30 days on ½ Lep media with 10 mg/l IAA (B), ½ Lep media with
20 mg/l IAA (C), 1/3 MS NH4NO3 free media with 1mg/l IAA (D)
18
program had abnormal morphology and signs of hyperhydricity after one month.
95 % of shoots on media with 10 or 20 mg/l IAA (fig. 21 B, C) became fragile,
swelled with curled and glassy leaves and callus growing along the stems. In
NH4NO3-free rooting medium with 1 mg/l IAA lower leaves of plants were
hyperhydrified but the top leaves were green and started to grow (fig. 21 D).
Table 6 shows that rooting of shoots in liquid media was very poor independently of
the culture medium, type of hormone and concentration. Rooting media with
different combination of auxin and cytokinin did not result in root formation,
besides plants were hyperhydrified in all these variants. To solve the problem of
hyperhydricity and unsuccessful rooting, plants were exposed to rooting medium with
20 mg/l IAA for 3 days and thereafter, explants were transferred to a hormone-free
liquid medium for root expression. However, this approach did not result in root
appearance in A. manshuriensis and problem with hyperhydric plants were not solved.
Rooting was not improved after splitting the shoots and cultured on 1/3 Lep or
1/3 MS media with low concentration (1mg/l) of IBA or 2,4-D.
Table 6. Development of rooted plants on liquid rooting media in TIS
Type of media
Hormone
Number
Hyperhydricity
rooted plants
Concentration, mg/l
Type
Auxin in high concentration
1/3 Lep
IAA
20
½ Lep
IAA
20
1/3 MS
IAA
20
1/3 Lep
IAA
10
1/3 MS
IAA
10
Auxin in high concentration during short period
1/3 Lep
IAA
20 (for 3 days)/0
1/3 MS
IAA
20 (for 3 days)/0
Combination of auxin and cytokinin
1/3 MS
IBA + kinetin
2+2,5
1/3 MS
IBA + BAP
1,5+2
1/3 MS
IBA + NAA
2+2
1/3 MS
kinetin + NAA
2,5+2
1/3 MS
IAA + BAP
1+0,1
Auxin in low concentration
1/3 Lep
2,4-D
1
1/3 MS
2,4-D
1
1/3 Lep
IBA
1
1/3 MS
IBA
1
1/3 Lep
IAA
1
1/3 MS
IAA
1
1/3 Lep + MgSO4
IAA
1
1/3 MS + MgSO4
IAA
1
Lep NH4NO3-free
IAA
1
MS NH4NO3-free
IAA
1
+
+
+
+
+
0
0
0
0
0
+
+
0
0
+
+
+
+
+
0*
0*
0*
0*
0
+
+
+
+
+
+
–
–
+
+
0*
0*
0*
0*
1 out of 52
1 out of 50
0
3 out of 21*
1 out of 52
0
* – splitting of basal ends of the shoots
19
Roots occasionally developed on shoots cultured in some media only with 1 mg/l
IAA. One rooted plant out of 50 was developed on 1/3 MS medium and one out of
52 – on 1/3 Lep medium. The number of rooted plants on 1/3 MS medium
increased (3 rooted plants out of 21) after increasing MgSO4 concentration in
medium and splitting the basal ends of shoots. Although not all of rooted plants
were vigorous, only 2 of them had leaves and roots at the same time (fig. 22). The
plants on 1/3 Lep medium with the same amount of hormone and extra MgSO4
were pail and did not produce any roots. Plants cultivated on either 1/3 MS or 1/3
Lep media with low concentration of IAA were not hyperhydrified. The number of
roots per plant cultivated in TIS was 1-3. The maximum length was 17 mm,
whereas usually the length was 3-5 mm after 4 weeks in liquid media.
Fig. 22. Rooted plants on 1/3 MS liquid media with 1 mg/l IAA and
extra MgSO4. The basal ends of shoots were split.
Ex vitro rooting of shoots
In sterile soil boxes
In vitro shoots placed in sterile boxes with soil supplemented with rooting media with
5 or 10 mg/l IAA started to grow in two weeks. They had green leaves and were
higher than plants on medium with 20 mg/l IAA. These boxes with two treatments
were removed from the experiment because contamination appeared in 2 weeks. In
the box with 20 mg/l IAA 12% of plants survived in 4 weeks but leaves were brown,
plants suffered and did not grow. The sterile soil boxes were kept in growth
chamber for one week and then placed in greenhouse. Probably high humidity or
mist system in greenhouse conditions made hydrophobic filters of plastic bag wet so
they could not keep sterile conditions inside the box and contamination appeared.
In soil
After ex vitro rooting of shoots in soil supplemented with different rooting media
(fig. 23) the percentage of rooted plants (fig. 24) without any treatment was 27 %
and with 1/3 Lep media supplemented with 5 or 20 mg/l IAA in soil – slightly
higher, 30 and 33% respectively. 10 mg/l IAA treatment in 1/3 Lep medium
20
A
B
C
Fig. 23. Plants after 3 days (A), 1 month (B), 2 months (C) after ex vitro rooting in soil
supplemented with rooting media containing different auxin concentrations
resulted in 23% survival and 30% – in 1/3 MS medium. Dipping of shoots in
250 mg/l IAA solution for 20 minutes resulted in the highest percentage (45%) of
plant survival in 4 weeks when plants were kept in styrofoam boxes. If they were
kept outside styrofoam boxes and covered with plastic film the survival was 6%
after dipping and 40% without any treatment. After 2nd month the number of
acclimatized plants slightly decreased (not more than 5%).
% of rooted plants
60
in 1 month
in 2 month
45
30
15
0
Control 1
5 IAA
1/3 Lep
10 IAA 10 IAA
1/3 Lep 1/3
MSMS
in styrofoam box
20 IAA Dipping 1 Control 2 Dipping 2
1/3 Lep
under plastic film
Fig. 24. Percentage of ex vitro rooted plants after one and two month in soil
supplemented with rooting media (1/3 Lep or 1/3 MS) with different auxin
concentrations or after dipping into auxin solutions as pretreatment kept in
styrofoam boxes or under plastic film. Control = no treatment.
21
Acclimatization of in vitro rooted shoots
Plants from semi-solid media
Table 7 shows the percentage of survival, after 4 weeks ex vitro, of shoots
cultivated on different rooting media. Rooted plants without leaves did not survive
ex vitro no matter what rooting medium had been used. Wilted, pour developed
plants with roots had very low (4%) survival. Only 8-10% of partly green rooted
plants stayed alive after 4 weeks ex vitro. 60-70% of almost green rooted shoots
survived. However, green plants with well-developed roots had the highest
percentage of survival (80-95%). Green shoots without roots showed that they
could acclimatize and develop roots ex vitro but the percentage of survival depend
on shoot appearance that corresponded to the type of rooting medium they were
cultivated on before: 70% of green shoots with big callus cultivated in vitro on 1/3
MS medium with 1,5 mg/l IBA together with 2 mg/l BAP and 40% on MS with
1 mg/l IAA together with 0,1 mg/l BAP survived ex vitro. Green shoots without roots
cultivated on 1/3 MS or 1/3 Lep media with 1 mg/l IAA and extra MgSO4 had low
survival (15 and 16% respectively) because they had small callus before ex vitro
transfer. None of the green plants dipped for 2 min into 250 mg/l IAA solution and
cultured on low BAP media had callus, so they did not acclimatized ex vitro.
Table 7. The percentage of survival, after 4 weeks ex vitro, of different types of shoots
cultivated on different rooting media before ex vitro
Type of
media
1/3Lep 10mg/l
IAA
MS 1mg/l IAA +
0,1mg/l BAP *
1/3MS 1,5mg/l
IBA+2mg/l BAP *
1/3MS 1mg/l IAA
+ extra MgSO4
1/3Lep 1mg/l IAA
+ extra MgSO4
Dipping 2’ in IAA
+low BAP media
The percentage of survival of different types of shoots
Green plant
Green
2/3 green
1/2 green
1/3 green No leaves
without roots rooted plant rooted plant rooted plant rooted plant rooted plant
–
–
–
–
4
0
40
80
62
–
–
–
70
–
–
–
–
–
15
95
70
8
0
0
16
88
67
10
0
0
0
–
–
–
–
–
* – splitting of basal ends of the shoots
Green shoots with well-developed roots cultured on 1/3 MS or 1/3 Lep semi-solid
media with 1 mg/l IAA and extra MgSO4, were transferred to 3 different conditions:
a) tray with soil mixture 2 peat:1 perlite (v/v) (fig. 25 A); b) pots with the same soil
mixture (fig. 25 B) and c) peat plugs (fig. 25 C), were successfully acclimatized
(95%) but growth intensity was different in 4 weeks (fig. 25 D, E, F). Plants
22
cultivated in tray were 1,5 times higher than those in pots and 2,5 times higher
than those in plugs (fig. 25 G, H, I). They also had 4 and 6 times bigger leaves
than those in pots and plugs respectively.
A
B
C
D
E
F
G
H
I
Fig. 25. Acclimatization of in vitro rooted plants in tray with peat:perlite mixture (A, D, G), in
pots with peat:perlite mixture (B, E, H) and in peat plugs (C, F, I) after 0 day (A, B, С) and in
30 days (D, E, F, G, H, I). A-F – top view, G-I – side view. G – plants from trays were
transferred to pots individually in 4 weeks for side view picture.
Plants from TIS
Hyperhydricity was a serious problem during culture of A. manshuriensis in TIS.
Despite this, abnormal and hyperhydrified shoots cultured on rooting media in TIS
were planted ex vitro (fig. 26). All hyperhydrified plants declined in 2 weeks no
matter of medium type they were cultivated in TIS before ex vitro acclimatization.
70% of healthy shoots without roots cultured in MS medium with 1 mg/l IAA in TIS
Fig. 26. Hyperhydrified shoots of cultivated on
rooting media in TIS planted ex vitro
23
did not recover and declined in two-three weeks after transferring to soil and in 6
weeks only 5% of plants survived. None of the rooted plants without leaves from
that medium stay alive in 4 weeks. If plantlets cultured in TIS had roots and leaves
at the same time they survived to 50%.
Chemical analysis for development of rooting media
Since micropropagated shoots of Aristolochia manshuriensis generally dropped the
leaves during rooting probably due to high sensitivity to auxins and NH4NO3 we
decided to analyze and compare mineral nutrients of leaves of in vitro grown shoots
and field grown plants to develop new rooting media in order to get green vigorous
rooted plants in vitro.
Chemical analysis
Table 8 shows the results from chemical element analysis of field and in vitro grown
plants. The content of the main macro elements nitrogen (N), phosphorus (P),
potassium (K), magnesium (Mg), calcium (Ca), sulfur (S) in plants grown in vitro
were less than in field grown plants. The content of Mg and P were almost twice less
and Ca – more than 4 times less in in vitro plants compared to field grown plants.
Table 8. Chemical analysis of shoots from field and in vitro grown plant material
Fe
In vitro grown shoots,
mg/kg DW
130
Field grown plant,
mg/kg DW
60
Al
49
19
B
120
39
Mo
22
7,3
Cu
1,4*
12
P
2300
5000
S
1900
2300
Zn
88
18
Cd
<0,7
<0,2
Mn
160
24
Ni
2*
0,5*
Na
970
76
Mg
1000
1700
Ca
2500
11000
K
22000
33000
Si
244
563
Total N
27400
35900
Element
24
Media development for rooting
Based on chemical analysis a new medium for rooting was developed. The
revised medium consisted of basal Lep macronutrients with increased
concentrations of KH2PO4, MgSO4, MS micronutrients, MS vitamins and lack of
NH4NO3. Table 9 shows the revised Lep* macronutrient compositions in comparison
with ordinary Lep and MS media.
Table 9. Macronutrient compositions of Lep, MS and the revised Lep* plant tissue
culture media
Macro salts
Ca(NO3)2
CaCl2
KNO3
NH4NO3
KH2PO4
MgSO4
Lep medium, mg/l
–
322
1900
1650
170
181
MS medium, mg/l
579
–
1800
400
270
176
Revised Lep* medium, mg/l
–
322
1900
–
400
800
Different concentrations of myo-inositol together with increased concentrations of
KH2PO4 and MgSO4 and lack of NH4NO3 were tested for improvement of
vigorous rooted shoots (table 10).
Table 10. Rooting and shoot development on new rooting media
Green shoot growth: ± little, + strong; callus development: – none
Type Concentration myoConcentration
of
of NH4NO3, inositol,
of IAA, mg/l
media
mg/l
mg/l
Lep media
Lep
0
100
Lep
0
200
Lep
0
600
1
Revised Lep* media
Lep*
400
200
Lep*
0
100
Lep*
0
200
Green
shoot
growth
Callus
development
%
of rooted
plants
±
±
±
–
–
–
0
11
0
+
+
+
–
–
–
6
11
22
Myo-inositol at 100 and 600 mg/l did not stimulate rooting in NH4NO3-free Lep
media supplemented with 1 mg/l of IAA, whereas at 200 mg/l of myo-inositol 11%
of rooted plants were obtained after 4 weeks of cultivation. The elevated
concentration of K, P, Mg stimulated both the root development and vigorous of the
shoots in Lep media no matter of presence or absence of NH4NO3 in media or
myo-inositol concentration. The presence of NH4NO3 in revised Lep* medium with
200 mg/l of myo-inositol led to achieve 6% of rooted plants. The lack of NH4NO3 in
revised Lep* medium improved the percentage of vigorous rooted plants: 11% –
with 100 mg/l myo-inositol and 22% – with 200 mg/l myo-inositol.
25
Conclusion
A. manshuriensis, as well as other members of the family Aristolochiaceae, have
not been propagated in liquid media in TIS before. The biomass fresh weight, the
growth parameters and the morphology of shoots produced in the bioreactor varied
for the different immersion cycles. When immersion applied twice per 24 h with
hourly 4 min ventilation, hyperhydric symptoms appeared in 90% of the shoots. A
review of the literature reveals that the immersion cycles used vary substantially
depending on the species involved, and on the micropropagation processes and
temporary immersion culture systems used (Etienne and Berthouly, 2002). It is
known that several cultural and physical factors in a typical in vitro environment are
decisive factors related with hyperhydricity. Theoretically, hyperhydricity can be
eliminated by changing hormone concentration or controlled in temporary
immersion systems by adjusting the immersion times. Propagation of Aristolochia
manshuriensis in a temporary immersion Plantform bioreactor was improved by
optimizing the frequency of immersions and duration of ventilation together with
BAP concentration. It was demonstrated that changing the immersion frequency
from 2 times to 1 or 6 times per 24 hours together with increasing ventilation
duration from 4 min to 10 min proved the most beneficial conditions of plant growth
in TIS together with low BAP concentration, resulting in 60% healthy plants but did
not prevent hyperhydricity symptoms completely. Decreasing the frequency of
immersions (1 time per 33 or 48 hours) improved quality of plants but the growth
was very slow. Frequency and duration of immersions probably influence the
success of the micropropagation process by influencing nutrient and water uptake
and consequently growth of the cultured material.
Different types of media and hormone concentrations were tested in order to
improve rooting ability in vitro but root formation was accompanied with shoot
wilting. In vitro study of developing roots of A. manshuriensis plants with different
hormone concentration and macro salts showed that 1/3 Lep medium
supplemented with 1 mg/l IAA and extra 600 mg/l MgSO4 allowed to get 70%
rooted plants on semi-solid medium but only 9% of rooted plants were vigorous
and had green leaves. 1/3 MS medium supplemented with 1 mg/l IAA and
600 mg/l MgSO4 resulted in root development in both semi-solid and liquid media,
but the percentage of rooted plant was not high (37% and 14% respectively).
However, only 9% of all plants were vigorous and have roots at the same time in
these media. Medium supplemented with 10 mg/l IAA showed 42% of rooted
plants on semi-solid medium but the shoots were weak and wilted. Using 1/3 Lep
medium with low concentration of IAA (1 mg/l) together with BAP (0,1 mg/l)
allowed to get green rooted plants but the percentage of root development was
low (8%) and plantlets formed large basal callus. Splitting of basal ends improved
root development in liquid and semi-solid media.
26
The percentage of rooted plants in TIS was lower than in semi-solid media
maybe because of serious hyperhydricity problem. 17% of shoots cultivated on
semi-solid NH4NO3-free MS medium supplemented with 1 mg/l IAA produced
roots, whereas 0% in liquid medium. The degree of abnormality depended on
type and concentration of hormone. The higher auxin concentration the more
serious problem with hyperhydricity, so immersion and ventilation programs for
rooting in TIS have to be improved.
Acclimatization ex vitro depended on the quality of rooted plants. Although
rooting in vitro was difficult, 95% of the plantlets from semi-solid media and 50%
from liquid media were successfully acclimatized when plants were with green
leaves and roots at the same time. Plants cultivated in semi-solid rooting media
survived better ex vitro than plants from liquid media, probably because of
hyperhydricity problem in TIS. The acclimatization of the plants from TIS resulted
in only 5% of survival ex vitro. In the literature (Pospíšilová et al., 1999) there is
information that the abnormalities in morphology, anatomy and physiology of
plantlets cultivated in vitro can be repaired after transfer to ex vitro conditions but
this did not apply for A. manshuriensis plants cultivated in TIS.
Ex vitro rooting by dipping of micropropagated shoots in 250 mg/l IAA solution for
20 min and transfer to soil led to 35% rooted plants in two month when the pots
were kept in styrofoam box. 33% of green vigorous micropropagated shoots
without any pre-rooting treatment could survive ex vitro and start to grow within 2
months. Ex vitro rooting in sterile soil boxes did not give positive results.
Rooting media based on differences in chemical analysis between field and in vitro
grown plants led to improve rooting. Semi-solid NH4NO3-free Lep medium
containing Lep macronutrients with elevated concentrations of KH2PO4 and
MgSO4 up to 400 and 800 mg/l respectively, and MS micronutrients, MS vitamins
supplemented with 1 mg/l IAA, 200 mg/l of myo-inositol resulted in 22% green
vigorous Aristolochia plants with roots and with 90% survival. Thus successful ex
vitro adaptation of rooted Aristolochia manshuriensis plants can be achieved in
this medium.
27
Literature
Hansen E., Rudin L. Nordström B. 1999. Odling av plantskoleväxter: 55, 179,
186. Natur och Kultur/LTs förlag, Borås. ISBN: 91-27-35236-6.
Hedman Y. 2005. Studies of root formation of micropropagated shoots in vitro
and cuttings from light treated mother plants ex vitro of Manchurian Dutchman’s
pipe (Aristolochia manshuriensis) Master thesis
Linsmaier E.M. Skoog F. 1965. Organic growth factor requirement of tobacco
tissue culture. Physiol. Plant. 18, 100-127.
Murashige T. Skoog F. 1962. A revised medium for rapid growth and bioassays
with tobacco cultures. Physiol. Plant. 15: 473-497.
Pospíšilová J., Tichá I., Kadleček P., Haisel D., Plzáková Š. 1999.
Acclimatization of micropropagated plants to ex vitro conditions. Biol. Plant. 42
(4): 481-497.
Quoirin M., Lepoivre P. 1977. Etude de milieu adaptes aux cultures in vitro de
Prunus. Acta Hort. 78: 437-442.
Svensson M. 2000. Effect of irradiance level during in vitro propagation of
Aristolochia manchuriensis. Acta Hort. 530: 403-408.
www.plantform.se
28
Light impact on shoot growth,
quality and rooting of
Aristolochia manshuriensis in vitro
Ph.D. T. Kuznetsova
Supervisor: Prof. M. Welander
SLU, Swedish University of Agricultural Sciences,
Department of Plant Breeding and Biotechnology
Alnarp, 2012
1
Contents
Introduction ........................................................................................................................ 3
Material and methods ....................................................................................................... 4
Plant material ................................................................................................................. 4
Growth conditions .......................................................................................................... 4
Light treatments ............................................................................................................. 4
Measurements ............................................................................................................... 5
Pigment analysis............................................................................................................ 5
Results ................................................................................................................................ 7
Light impact on shoot growth and quality .................................................................. 7
Growth parameters .................................................................................................... 7
Pigment analyses ..................................................................................................... 10
Light impact on shoot and root development .......................................................... 12
Rooting ....................................................................................................................... 12
Pigment analyses ..................................................................................................... 14
Conclusion ....................................................................................................................... 16
Literature .......................................................................................................................... 17
2
Introduction
The ornamental plant Aristolochia manshuriensis Kom. is difficult to root in vitro
(Svensson, 2000, Hedman, 2005). Svensson (2000) noted that high light intensity
improved rooting of this species in vitro, but the number of rooted plants was still
very low. Recently several papers were published (Wu and Lin, 2012, Shin et al.,
2008, Jao and Fang, 2004 etc.) regarding positive impact of light-emitting diodes
(LEDs) on growth and root development of plants cultured in vitro. To our current
knowledge there are no studies on impact of LEDs on A. manshuriensis. For this
reason we investigated the effects of monochromatic and polychromatic lights on
shoot growth and root development in vitro by using LEDs and plant growth
fluorescent lamps (PGF). The light impact on synthesis of different photosynthetic
pigments was analyzed additionally.
3
Material and methods
Plant material
Shoot cultures of Aristolochia manshuriensis Kom. kindly provided by Lars
Sommer (Vitroform, Denmark) were used in all experiments.
Growth conditions
In vitro shoots were cultured either on growth or rooting media (table 1) based on
Lep (Quoirin and Lepoivre, 1977) macronutrients, MS (Murashige and Skoog, 1962)
micronutrients, MS vitamins or MS medium containing MS macronutrients, MS
micronutrients, MES buffer and LS (Linsmayer and Skoog, 1965) vitamins
supplemented with 3% sucrose and 7% Bacto agar. Rooting media were
supplemented with 2 g/l activated charcoal. Different concentrations of IAA alone or
together with BAP were used in rooting media. Full and 1/3 strength of Lep rooting
media, lack of NH4NO3 and extra 100 mg/l of myo-inositol were tested as well. The
pH was adjusted to 5,5 in Lep and 5,7 in MS media with NaOH before autoclaving
for 20 min at 120°C. Nine (5 mm) segments without leaves for shoot production and
six (20-25 mm) shoots for root initiation were placed per 0,4 l plastic jars (Styrolux
Thermoplastic) with 62,5 ml of fresh semi-solid medium. The shoots were kept in a
phytotron chamber for a month under different light treatments (fig. 1).
The temperature outside the boxes was kept at 23°C during day and 18°C at night
and relatively humidity was 45%.
Table1. Combinations of hormones and their concentration in Lep or MS media for shoot
growth and root development under different light treatments
No
Type of media
Growth media
Lep
MS
Rooting media
1. 1/3 Lep+charcoal
2. 1/3 Lep+charcoal
3. Lep NH4NO3 free+myo-inositol+charcoal
4. Lep NH4NO3 free+myo-inositol+charcoal
5. 1/3 Lep NH4NO3 free+charcoal
type
Hormone
concentration, mg/l
BAP
BAP
0,15
0,15
IAA
IAA+BAP
IAA
IAA
IAA
1
1+0,1
5
1
1
Light treatments
Light-emitting diodes (LEDs) (fig. 2) including 100% white, 70% red + 30% blue,
100% blue, 100% red and fluorescent lamps (PGF) at high (HL) and low (LL) light
intensity were used in the experiments. Six light environments were established by
using commercially available lamps. To avoid interference between the light
4
a
b
a
b
c
d
c
d
Fig. 1. Shoots in plastic jars under different
LED treatments
Fig. 2. Light-emitting diodes (LEDs): white (a),
blue and red (b), blue (c), red (d)
treatments in the same phytotron chamber, each light compartment
(70x70x200 cm) was divided by white-black plastic film, with white side inside. The
light intensity of all LEDs and PGF treatments was 90 μmol·m-2s-1 except low light
PGF at 33 μmol·m-2s-1 and at a photoperiod of 18 h day-1. The different light
parameters are given in table 2.
Table 2. Light treatments
LEDs
Parameters
and units
Wave length, nm
Light intensity,
μmol·m-2s-1
PGF
white
red
blue
(W)
(R)
(B)
red:blue high light low light
7:2
(V)
(HL)
(LL)
430-730
620
469
620-469 430-730 430-730
90
90
90
90
90
33
Measurements
The shoot height, shoot number, fresh weight (FW) and dry weight (DW) (on
freeze-dried material), petiole length and leaf number per shoot were measured
on in vitro produced shoots cultivated on different growth media for 30 days under
LEDs and fluorescent lights. On rooting media the percentage of rooted shoots,
callus growth and green shoots were recorded after one month.
Pigment analysis
Only upper leaves fully exposed to light from shoots cultured on either growth or
rooting media (except medium 1) under different light sources were collected for
pigment analysis. Sample preparation and chlorophyll extraction was conducted in
5
dark to avoid possible photo bleaching. Leaves were freeze-dried and ground to
powder. 0,05 g of lyophilized powder was extracted with 1 ml ethyl acetate/ethanol
80/20. After ultrasonic bath (about 10 min), heating bath at 60°C (60 min) and
centrifugation (10 min at 7500g), the supernatant was collected and used for a highperformance liquid chromatography (HPLC). Chlorophyll a and b as well as
β-carotene, lutein and xanthophyll content were determined. The pigments were
analyzed using HPLC system with a 250 mm x 4,6 mm i.d., 5 µm particle size,
Phenomenex Silica column according to a method modified by Panfili (2003, 2004).
6
Results
Light impact on shoot growth and quality
Growth parameters
The appearance of in vitro shoots cultivated on Lep growth medium cultured under
different light treatments for 30 days did not show clear differences (fig. 3). However
the shoot height (fig. 4) increased with light intensity from 33 μmol·m-2s-1 to
90 μmol·m-2s-1 under fluorescent light and resulted in the highest value (2,2 cm)
among all light treatments. Shoots under fluorescent light also developed more leaves
than under LED lights (fig. 5). 80% of the shoots had minimum 2 leaves under all light
regimes. The highest percentage of shoots with more than 3 leaves was obtained
under HL PGF (67%). Fluorescent light also stimulated development of leaf 5 and 6.
W-LED
B-LED
V-LED
R-LED
HL-PGF
LL-PGF
Fig. 3. Shoots cultured on Lep growth media for
30 days under 6 light treatments
7
% of shoots with different leaf number
2.5
Shoot height, cm
2
1.5
1
0.5
0
100
90
80
70
60
50
40
30
20
10
0
Lep MSLep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W R R B B V V
Media type
Light treatment
Fig. 4. Shoots height after 30 days on Lep
or MS media with 0,15 mg/l BAP under 6
light treatments
1
2
3
4
5
6
Leaf number
Fig. 5. Percentage of shoots developed
different leaf number (1–6) under 6 light
treatments after 30 days in culture on MS or
Lep media with 0,15 mg/l BAP
1.4
2.5
1.2
Length of petiole, cm
2
Shoot number
Lep MSLep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W R R B B V V
1.5
1
0.8
0.6
1
0.4
0.5
0.2
0
0
Lep MSLep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W R R B B V V
Media type
Light treatment
Fig. 6. Shoot number after 30 days in
culture on MS or Lep media with 0,15 mg/l
BAP under 6 light treatments
Lep MSLep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W R R B B V V
Media type
Light treatment
Fig. 7. Length of petiole after 30 days in
culture on MS or Lep media with
0,15 mg/l BAP under 6 light treatments
Shoot number (fig. 6) and length of petiole (fig. 7) did not differ significantly under
the different light regimes.
8
Figure 8 A and B show that the highest value of FW (2,2 g) and DW (0,4 g) were
obtained in shoots grown under HL-PGF. The dry matter content also increased
from 14-18% with increasing light intensity from 30 μmol·m-2s-1 to 90 μmol·m-2s-1
(fig. 8 C). The highest dry matter content in shoots grown under LED lights was
observed under white light.
3.0
A
FW, g
2.0
1.0
0.0
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W R R B B V V
DW, g
B
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W R R B B V V
20
C
DW/FW, %
16
12
8
4
0
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W R R B B V V
Media type and
Light treatment
Fig. 8. Fresh weight (A), dry weight (B), dry matter (C) of shoots
cultivated on MS or Lep media with 0,15 mg/l BAP for 30 days
under 6 light treatments
9
Pigment analyses
The concentrations of chlorophyll a in leaves of shoots cultured on the different
growth media ranged from 6000-7000 µg/g DW (fig. 9 A) and chlorophyll b around
2000 µg/g dW (fig. 9 B) under all light treatments, except for HL-PGF. Shoots under
HL-PGF had the lowest concentrations of chlorophyll a and b. The ratio of
chlorophyll a and b was similar in all treatment, except under HL-PGF treatment in
Lep media (fig. 9 C). Light abbreviations see table 2.
A
Chlorophyll a, µg/g dW
10000
8000
6000
4000
2000
0
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W V V R R B B
B
Chlorophyll b, µg/g dW
3000
2500
2000
1500
1000
500
0
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W V V R R B B
C
chlorophyll a /b ratio
5
4
3
2
1
0
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W V V R R B B
Media type and
Light treatment
Fig. 9. Chlorophyll a (A) and b (B) content (µg/g dW) and
chlorophyll a/b ratio (C) in leaves of shoots cultured on MS or Lep
media with 0,15 mg/l BAP for 30 days under 6 light treatments
10
Leaves of shoots cultured under HL-PGF had the lowest content of lutein (fig. 10 A).
Shoots grown under W-LED and B-LED on Lep medium had also slightly lower lutein
content compared to the other light treatments. Figure 10 B and C show no significant
differences in the concentrations of β-carotene (260–300 µg/g dW) and xanthophyll
(200–250 µg/g dW) between media and light treatments.
A
Lutein, µg/g dW
1000
800
600
400
200
0
B
β-carotene, µg/g dW
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W V V R R B B
400
350
300
250
200
150
100
50
0
C
Xanthophyll, µg/g dW
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W V V R R B B
400
350
300
250
200
150
100
50
0
Lep MS Lep MS Lep MS Lep MS Lep MS Lep MS
LL LL HL HL W W V V R R B B
Media type and
Light treatment
Fig. 10. β-carotene (A), lutein (B) and xanthophyll (C) content
(µg/g dW) in leaves of shoots cultured on MS or Lep media with
0,15 mg/l BAP for 30 days under 6 light treatments
11
Light impact on shoot and root development
Rooting
In vitro shoots cultured under different light treatments for 30 days on different
rooting media showed different appearance (fig. 11). Shoots on media 3 and 4
Media 3
Media 4
Media 5
Media 2
W
V
R
B
HL
LL
Fig. 11. Shoot appearance in response to 6 light treatments after 30 days in culture on 4 different root media
12
(table 3) with full strength of Lep macro nutrients were more vigorous and
developed better than on media 5 and 2 (table 3) with 1/3 strength of Lep macro
nutrients no matter of hormone concentration. Besides plants cultured on medium
5 with 1 mg/l IAA looked better that on medium 2 supplemented with IAA together
with BAP. Growth of leaves and their development were also influenced by
light treatment better on full strength media and depended on light treatment. Plants
grown on media 3 and 4 under LL-PGF treatment were greener and more vigorous
than in other light treatments.
Table 3 shows the impact of 6 light treatments on shoot and root development
cultured on 5 different rooting media. Root development was very poor and was
achieved only on medium 1 under R-LED (4%), on media 2 and 5 under LL-PGF
(8 and 4% respectively) and on medium 3 under B-LED (4%) and V-LED (4%).
Table 3. Shoot and root development on rooting media under different light treatments
Hormone
Media
Light
Type of media
code
treatment
Type
Concentration, mg/l
LL
HL
W-LED
1/3Lep
1
IAA
1
+charcoal
V-LED
R-LED
B-LED
LL
HL
W-LED
1/3Lep
2
IAA+BAP
1+0,1
+charcoal
V-LED
R-LED
B-LED
LL
HL
Lep NH4NO3
W-LED
free
3
IAA
1
+myo-inositol V-LED
+charcoal
R-LED
B-LED
LL
HL
Lep NH4NO3
W-LED
free
4
IAA
5
+myo-inositol V-LED
+charcoal
R-LED
B-LED
LL
HL
1/3Lep
W-LED
5 NH4NO3 free
IAA
1
V-LED
+charcoal
R-LED
B-LED
Green
% of rooted
Callus
shoot
plants
±
–
0
±
–
0
±
–
0
±
–
0
4
±
–
±
–
0
8
±
+
±
+
0
±
+
0
±
+
0
±
+
0
±
+
0
+
–
0
+
–
0
+
–
0
4
+
–
+
–
0
4
+
–
+
–
0
+
–
0
+
–
0
+
–
0
+
–
0
+
–
0
4
±
–
±
–
0
±
–
0
±
–
0
±
–
0
±
–
0
13
Only one root was usually developed per shoot with a root length of 1-2 mm after 30
days of culture (fig. 12 A, B), except one formed on medium 1 under R-LED with a root
length of 10 cm (fig. 12 C).
A
B
C
Fig. 12. Rooted plants developed under different light treatments and media: A – LL-PGF on
medium 2, B – B-LED on medium 3, C – R-LED on medium 1 (bar = 1 cm)
Pigment analyses
Shoots grown on media 3 and 4 had the highest content of chlorophyll a and b
(fig. 13 A, B) under LL-PGF and B-LED. Shoots grown on medium 2 had the highest
chlorophyll a content under R-LED and also higher content under HL-PGF, W-LED
and V-LED compared to media 3, 4 and 5 (fig.13 A). The content of chlorophyll b
was only higher under R-LED (fig.13 A, B). The content of β-carotene was also
highest in media 3 and 4 under LL-PGF and B-LED and on medium 2 β-carotene
was slightly higher under W-LED and V-LED (fig. 13 C). The highest content of lutein
was found in medium 2 under V-LED and R-LED (fig. 13 D). The highest content of
xanthophyll was found also in media 3 and 4 under all light treatments, except
R-LED (fig. 14). The pigment content in shoots grown on medium 5 was generally
lower under all light treatments (fig. 13, 14). The ratio between chlorophyll a and b
(fig. 15) was higher in media 5 and 2 compared with other media and light treatments
and the highest ratio was observed under B-LED.
The overall low pigment content in medium 5 can be explained by the low
N-content due to reduced Lep macronutrients to 1/3 and lack of NH4NO3. The
shoots were also less vigorous compared with media 3 and 4 especially under
R-LED, B-LED, HL-PGF and LL-PGF. The shoot appearance in medium 2 was
very similar to medium 5 although the pigment content was much higher. This can
be explained by full NH4NO3 and addition of 0,1 mg/l BAP in the medium.
Shoot appearance and pigment content were very similar in media 3 and 4
indicating that the higher auxin concentration in medium 4 had no influence on
either pigment concentration or shoot appearance. However the most developed
shoots were obtained on medium 3 under LL-LED also with the highest content of
chlorophyll a and b as well β-carotene.
14
B
A
4000
12000
3
4
5
2
Chlorophyll b, µg/g dw
Chlorophyll a, µg/g dw
10000
8000
6000
4000
3
3500
2000
5
2
3000
2500
2000
1500
1000
500
0
0
LL
HL
W-LED V-LED R-LED B-LED
LL
HL
Light treatment
W-LED V-LED R-LED B-LED
Light treatment
C
D
1200
600
3
4
5
3
2
4
5
2
1000
Lutein, µg/g dw
500
β-caronine, µg/g dw
4
400
300
200
800
600
400
200
100
0
0
LL
HL
LL
W-LED V-LED R-LED B-LED
HL
W-LED V-LED R-LED B-LED
Light treatment
Light treatment
Fig. 13. Chlorophyll a (A), b (B) content, β-carotene (C) and lutein (D) (µg/g dW) in leaves of
shoots cultured on 4 different root media for 30 days under 6 light treatments
8
350
3
4
5
2
Chlorophyll a/b ratio
Xanthophyll, µg/g dw
250
200
150
100
50
3
7
300
4
5
2
6
5
4
3
2
1
0
0
LL
HL
W-LED V-LED R-LED B-LED
Light treatment
Fig. 14. Xanthophyll content (µg/g dW) in
leaves of shoots cultured on 3 different root
media for 30 days under 6 light treatments
LL
HL
W-LED V-LED R-LED B-LED
Light treatment
Fig. 15. Chlorophyll a/b ratio in leaves of
shoots cultured on 4 different root media
for 30 days under 6 light treatments
15
Conclusion
The study of different lights on shoot growth of Aristolochia manshurensis in vitro on
semi-solid medium showed that fluorescent light at 90 μmol·m-2s-1 was beneficial for
growth of shoots cultured on Lep medium supplemented with 0,15 mg/l BAP. This
high fluorescent light resulted in increased shoot height, FW, DW and number of
leaves. The positive influence on the different growth parameters was not reflected
by higher pigment content. Instead the opposite relation was found. The highest
concentrations of all analyzed pigments were obtained under LL-PGF and the LED
light treatments with the lowest values on growth parameters.
Studies on the impact of lights on shoot quality and root development showed that
rooting of shoots was very poor (4–8%). Earlier rooting experiments on Aristolochia
shoots showed that the best rooting (70%) was obtained on medium with 1/3 of Lep
macronutrients with 1 mg/l IAA and extra 600 mg/l MgSO4. However more than 50%
of the rooted shoots dropped their leaves and were difficult to acclimatize. Only 9%
of all plants were vigorous and have roots at the same time. Also in this study shoots
on 1/3 of Lep macronutrients (media 2 and 5) were less green and vigorous no matter
of light treatment. However the rooting frequency was not better compared to full
strength Lep macronutrients without NH4NO3. On NH4NO3-free medium with
1 mg/l IAA (medium 3) rooting was obtained under V-LED and B-LED but no rooting
on medium 4 with a higher auxin concentration (5 mg/l) indicating an auxin optimum
for rooting. Interesting is that the highest rooting was obtained (8%) on medium with
1 mg/l IAA together with 0,1 mg/l BAP (medium 2) under LL-PGF with lowest pigment
content of this medium.
We can conclude that there is no positive correlation between pigment content and
growth parameters. Also different LED-light treatments did not result in better rooting.
Fuernkranz (1990) stated that both intensity and spectral quality of light significantly
affected root formation and growth. However this was not obtained for Aristolochia,
probably the studied light intensity was not optimal for root formation of this species.
16
Literature
Fuernkranz H.A., Nowak C.A., Maynard C.A. 1990. Light effects on in vitro
adventitious root formation in axillary shoots of mature Prunus serotina. Physiol.
Plant., 80: 337–341.
Hedman Y. 2005. Studies of root formation of micropropagated shoots in vitro and
cuttings from light treated mother plants ex vitro of Manchurian Dutchman’s pipe
(Aristolochia manshuriensis). Master thesis.
Jao Ruey-Chi, Fang Wei. 2004. Growth of potato plantlets in vitro is different when
provided concurrent versus alternating blue and red light photoperiods. Hort
Science. 39 (2): 380–382.
Linsmaier E.M. Skoog F. 1965. Organic growth factor requirement of tobacco
tissue culture. Physiol. Plant. 18, 100-127.
Murashige T. Skoog F. 1962. A revised medium for rapid growth and bioassays
with tobacco cultures. Physiol. Plant. 15: 473–497.
Panfili 2003. Agric. Food Chem. 51: 1322–1327.
Panfili 2004. Agric. Food Chem. 52: 6373–6377.
Quoirin M., Lepoivre P. 1977 Etude de milieu adaptes aux cultures in vitro de
Prunus. Acta Hort. 78:437–442.
Shin Kong Sik, Murthy Hosakatte Niranjana, Heo Joeng Wook, Hahn Eun Joo,
Paek Kee Yoeup. 2008. The effect of light quality on the growth and development
of in vitro cultured Doritaenopsis plants. Acta Physiol Plant. 30: 339-343.
Svensson M. 2000. Effect of irradiance level during in vitro propagation of
Aristolochia manchuriensis. Acta Horticulturae 530: 403–408.
Wu How-Chiun and Lin Chun-Chin, 2012. Red Light-emitting diode light irradiation
improves root and leaf formation in difficult-to-propagate Protea cynaroides L.
planlets in vitro. Hort Science. 47 (10): 1490–1494.
17